In recent years, a protein which can be utilized in medicaments has been extensively tried with the use of the gene recombinant technique. Since a protein having a great molecular size, various modifications such as addition of a sugar chain, and a protein of a subunit structure consisting of a plurality of polypeptide chains can not be responded in an expression system using microorganisms such as yeast and Escherichia coli as a host, a production system using animal cells as a host is used in many cases. Such a protein is produced using a mammal cell among the animal cells, in many cases. In the case where the protein is a secretion protein, since an objective protein is recovered in the culture supernatant, generally, a method for culturing a recombinant animal cell in a suitable medium, culturing the cell for a constant term and, thereafter, recovering the culture supernatant collectively (batch culturing), or a method for continuously performing extraction and addition of a suitable amount of a medium at an arbitrary time (perfusion culturing) is used. In any event, as the number of recombinant animal cells producing the objective secretion protein is increased, an accumulation (production) amount of a secretion protein in a culture is increased. Proliferation of a cell is classified into three terms of a logarithmic phase in which a cell is logarithmically proliferated, a stationary phase in which the number of cells is apparently constant, and a death phase in which cells die and the number of cells is decreased. In order to increase the production of the secretion protein, it is important to increase the cell density of the recombinant animal cell in the stationary phase as high as possible, and maintain the term as long as possible. Particularly, in the case of batch culturing, the recombinant animal cell is proliferated in the constant amount of the medium. Therefore, in order to increase the production amount of the secretion protein therein, various attempts have been tried to increase the cell density at the stationary phase as high as possible, and maintain the term as long as possible.
As an attempt to maintain the stationary phase long, from a viewpoint of growing, there has been adopted a method for improving the proliferating property to extend the stationary phase by devising a nutrient component such as improvement in a medium component and addition of a growth factor. In addition, as a culturing method, there is a method for extending the stationary phase long by supplementing a nutrient to a cell at the stationary phase at a suitable interval, to prevent nutrient depletion, such as a fed batch culturing method. A perfusion method is a method for performing this continuously. In order to increase the production amount of the objective protein, usually, such a growing method has been adopted. As a method which is different from the growing method, an attempt has also been tried to improve a host cell. For example, there has been tried a method for using an anti-apoptotic factor. This method is an attempt to express an anti-apoptotic factor gene in a recombinant animal cell producing a protein, imparting to the cell the ability of suppressing programmed cell death (apoptosis) generated by nutrient starvation, thereby extending the stationary phase.
According to Non-Patent Document 1, a mechanism for causing apoptosis is considered as follows. When a variety of cell death stimulations such as nutrient depletion are transmitted to a cell, the signal is transmitted to mitochondrion via various proteins including a transcription factor and a kinase. A mitochondrion having received the signal releases an apoptosis signal transmitting factor (AIF, cytochrome c etc.) into a cytoplasm. Cytochrome c binds to Apaf-1 (apoptosis activating factor-1) and pro-caspase-9 present in the cytoplasm to form a complex, and activates caspase-9. An activated caspase cascade cuts various intracellular or intranuclear substrates to induce a variety of morphological or biochemical changes (actin degradation, DNA fragmentation, chromosome condensation etc.) characteristic in apoptosis. As a factor suppressing such apoptosis, Bcl-2 (B cell lymphoma/leukemia 2) is well-known. The Bcl-2 gene was found as an oncogene which is seen in human follicular lymphoma at a high frequency. Currently, many family genes having a domain (BH1-4) having high homology with Bcl-2 are identified. In a family, there are a factor serving suppressively on apoptosis, and a factor serving promotively on apoptosis. As the suppressive factor, for example, Bcl-xL, Bcl-w, Mcl-1, A1, BHRF1, E1B-19K and Ced-9 are known, and it is thought that they inhibit signal transmission by inhibition of the aforementioned release of cytochrome c, or binding with Apaf-1 and procaspase-9. It is thought that the suppressive Bcl-2 family functions upstream of a caspase cascade.
On the other hand, there is also known a factor which acts downstream of the caspase cascade (inhibits the directly activity of the caspase) to exhibit the cell death suppressing effect. For example, a p35 protein of AcNPV (Autogropha californica nuclear polyhedrosis virus) belonging to a baculovirus family is cut as a substrate for caspase and, the fragment forms a stable complex with almost all caspases, so that activity thereof is inhibited. Therefore, various apoptosis can be suppressed. BmNPV (Bombyx mori nuclear polyhedrosis virus) closely-related to AcNPV also has a p35 gene. In addition, crmA of cowpox virus specifically binds to caspase-1-like protease or caspase-8,-10 to inhibit this, so that apoptosis can be suppressed. In addition, v-FLIP derived from herpesvirus has two DEDs (death effecter domains), and binds to an FADD (Fas-associating Protein with death domain), so that activation of caspase-8 is suppressed. Further, in many closely-related viruses including CpGV (Cidia pomonella granulosis virus) and OpMNPV (Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus) of a baculovirus family, a v-IAP (inhibitor of apoptosis) gene, the expression product of which directly inhibits caspase activity, has been identified, different from a p35 gene. Up to now, as a homologue of v-IAP, an IAP family having several kinds of BIRs (baculovirus IAP repeats) such as c-IAP1/hia-2, c-IAP2/hia-1, XIAP, NAIP, survivin, TIAP, Apollon, DIAP1, DIAP2, SfIAP and ITA has been identified in Drosophila and a mammal in addition to a virus.
An attempt has been tried to utilize the factor having anti-apoptosis activity, for example, a Bcl-2 family in cell culturing, but now, the effect on enhancement of a production amount of a protein has not been revealed regarding a Bcl-2 family. For example, when a Bcl-2 gene is introduced into a CHO cell producing a chimera antibody to express it, Tey B T et al. observed the viability extending effect, but an antibody production amount was not changed (see Non-Patent Document 2). Simpson N H et al. introduced a Bcl-2 gene into a hybridoma, but this did not also lead to increase in the antibody producing ability (see Non-Patent Document 3). Similarly, Kim N S, Lee G M et al. reported that, in an antibody-producing CHO cell in which a Bcl-2 gene is expressed in batch culturing, little change is recognized in an antibody production amount as compared with the case where the gene is not expressed (see Non-Patent Documents 4 and 5). On the other hand, they increased an antibody production amount by suppression of apoptosis inducing action possessed by butyric acid by Bcl-2 when sodium butyrate is added simultaneously and, consequently, enhancement of production amount potentiating action possessed by butyric acid (see Non-Patent Document 5).
In addition, they found out that, similarly, expression of Bcl-2 suppresses cell death due to a high osmotic pressure, and reported that a production amount can be increased by assisting antibody production potentiating effect due to a high osmotic pressure (see Non-Patent Document 6). These reports that, even when Bcl-2 exerts cell death suppressing effect, this is not directly involved in the effect of potentiating a production amount of a secretion protein such as an antibody. In addition, it was reported that expression of Bcl-2, Bcl-xL or E1B-19K acts towards reduction in cell proliferation (see Non-Patent Document 7). Similarly, MCL-1 which is a Bcl-2 family also improves in viability of a cell, but it does not influence on a signal of cell proliferation (see Non-Patent Document 8). Bcl-xL also improves cell viability, but there is a report that it does not contribute to improvement in a production amount of a secretion protein. For example, in a transgenic mouse in which a gene expressing Bcl-xL under control of an insulin promoter was introduced, Bcl-xL improved viability of a β cell, but decreased secretion expression of insulin due to glucose inducement rather than potentiated it (see Non-Patent Document 9). Similarly, when production of inflammatory cytokine such as TNFα due to LPS inducement was examined using a RAW264 macrophage cell in which Bcl-xL was expressed, a production amount was decreased (see Non-Patent Document 10). An E1B-19K gene which is similarly a Bcl-2 family was introduced into antibody-producing NS/O myeloma, but improvement in a production amount was not recognized (see Non-Patent Document 11).
Like this, although all of the methods using an anti-apoptotic factor derived from a Bcl-2 family such as Bcl-2, Bcl-xL and E1B-19K which have previously been tried can suppress cell death, and extend a stationary phase of a proliferation curve, a production amount is not increased as expected, in many cases. From these things, it is thought that the direct effect of potentiating a production amount of a protein is not present in these factors, or if any, the effect is exerted under the especial environment. On the other hand, regarding a factor having caspase inhibiting action, a representative of which is P35 of baculovirus, there is no report that a relationship with the production amount potentiating effect was investigated in a recombinant protein producing cell, much less, there is no report that there is the production amount potentiating effect in a recombinant secretion protein producing cell.
In the present invention, fibrinogen, ecarin and factor VIII are used as one example of a protein which is a subject of the present invention. Fibrinogen as one of blood coagulation factors plays a role in coagulating blood when a living body undergoes injury. The first function is to form a body of a thrombus called fibrin clot at an injured site, and the second function is to serve as an adhesive protein necessary for platelet aggregation. A blood concentration of fibrinogen is usually about 3 mg/ml, and this is the third highest next to albumin and immunoglobulin G. Fibrinogen is a macro glycoprotein consisting of a total six of polypeptides having each two of three different kinds of polypeptides called an α chain, a β chain and a γ chain. Individual molecular weights of polypeptides are such that the α chain has about 67000, the β chain has about 56000 and the γ chain has about 47500, and a molecular weight of fibrinogen which is an aggregate of them mounts to about 340000 (see Non-Patent Document 12). In fibrinogen in blood, there are heterogenous molecules due to existence of heterogenous polypeptides having different molecular sizes. For example, the existence of a heterogenous type called γ chain (or γB chain) in a γ chain has been reported, and it has been revealed that this is a polypeptide consisting of a total 427 of amino acid residues in which 20 amino acid residues are added to a 408-position of an amino acid sequence of the γ chain (see Non-Patent Document 13). In addition, there is a heterogenous type called αE is present also in the α chain, and it has been reported that this polypeptide has a total 847 of amino acid residues in which 236 amino acid residues are extended to a 612-position of an amino acid sequence of the α chain (see Non-Patent Document 14).
A fibrinogen preparation is effective in inhibiting severe bleeding by enhancing a fibrinogen concentration in blood by a method such as intravenous administration, and is used in improving the consumption state of a blood coagulation factor, for example, such as disseminated intravascular coagulation (DIC) in sepsis, or in replenishing therapy in congenital or acquired fibrinogen efficiency.
In addition, the fibrinogen preparation is also widely utilized as a tissue adhesive utilizing adherence of fibrin (see Non-Patent Document 15). This living body-derived adhesive utilizes gelation of fibrinogen in a living body, and is widely used in hemostasis, closure of a wound site, adhesion or suture reinforcement of a tissue such as nerve, tendon or vessel, and closure of air leakage in lung. In addition, in recent years, a preparation having enhanced convenience by attaching fibrinogen to a sheet of collagen etc. has been commercially available.
Currently, fibrinogen used as a medicament is prepared from human plasma, and examples of a problem thereof include 1) a risk of mixing in of an infective pathogen such as a virus causing pneumonia such as HAV, HBV, HCV, HEV and TTV, a virus causing immunodeficiency such as HIV, and abnormal prion causing CJD because of use of plasma collected from unspecified many humans, and 2) supply of plasma by blood donation in Japan and, consequently, a problem of stable supply in the future.
In order to overcome these problems, recombination of fibrinogen has previously been tried. For example, in Escherichia coli, a fibrinogen γ chain was successfully expressed in a bacterium, but there is no report that a functional fibrinogen molecule is produced by simultaneously expressing three proteins of an α chain, a β chain and a γ chain. In addition, also in an expression system using yeast, there was a report that secretion expression was successful at a certain time, but reproductivity was not finally confirmed, and the report was withdrawn (see Non-Patent Document 16). Like this, there has not been yet a report that fibrinogen was successfully expressed using Escherichia coli or yeast.
On the other hand, ecarin is a snake venom-derived protease which has been isolated and purified from Echis carinatus (Non-Patent Document 17), and is known to specifically convert prothrombin playing an important role in blood coagulation into activated thrombin. Thrombin used as a medicament is used as a hemostatic agent. It is used in bleeding from a small vessel, a capillary vessel and a parenchymal internal organ for which hemostasis is difficult by conventional ligation (e.g., bleeding accompanied with trauma, bleeding during operation, bone bleeding, bladder bleeding, bleeding after tooth extraction, nose bleeding, and bleeding from an upper digestive tract such as gastric ulcer). A current thrombin preparation is derived from bovine blood, or prepared from human plasma and, as a problem thereof, the preparation has the same problems as those of fibrinogen, such as 1) a risk of mixing in of an infective pathogen, and 2) stable supply in the future and, thus, thrombin obtained by the recombinant technique is desired. Upon preparation of such thrombin, it is difficult to produce thrombin as activated thrombin from the beginning, and it is necessary to produce prothrombin and, thereafter, activate the prothrombin using any enzyme. As an effective converting enzyme, ecarin is mentioned as a candidate, but ecarin is also derived from snake venom, has a problem of its supply and mixing in of an infective pathogen and, thus, recombination has been desired. In addition, since ecarin acts also on abnormal prothrombin biosynthesized in the absence of vitamin K, it is utilized in measuring a blood concentration of the abnormal prothrombin. However, only a small amount is purified from a snake venom, and it can not be used as a general reagent. That is, it is essential in a step of preparing a thrombin preparation made by the recombination technique to supply ecarin obtained by the recombination technique at a large amount and, also as a clinical diagnostic, high production has previously been desired for practical use of ecarin.
Factor VIII is an important coagulation factor which amplifies a reaction of activating factor X by activated factor IX about 200 thousands-fold in a blood coagulation reaction. When the factor VIII is deficient, there is a tendency of serious bleeding, and this is known as hemophilia A. Hemophilia A is a congenital bleeding disease based on deficiency in a blood coagulation factor VIII, is usually developed in a man, and an incidence rate is said to be one per 5000 to 10000 of male birth. A bleeding symptom begins at an infant stage and thereafter in many cases, and generally appears subcutaneously, intra-articularly, intramuscularly, hematurically, orally, or intracranially. When intra-articular bleeding is repeated, a joint disorder progresses, leading to chronic hemophilic arthrosis accompanied with limited movement of a joint. Therapy of hemophilia A is an intravenous injection of a factor VIII preparation in principle. Currently, both of a blood-derived factor VIII preparation and a recombinant preparation are commercially available, and a blood-derived preparation has a problem of a risk of mixing in of an infective pathogen and stable supply as described above. On the other hand, regarding a recombinant preparation, products are lacked, and supply becomes deficient, causing a social problem. For solving these problems, high production necessary for increasing production of the recombinant preparation has been desired.
In the case of fibrinogen, in an animal cell, expression has been tried using a BHK cell (see Non-Patent Document 18), a COS cell (see Non-Patent Document 19), or a CHO cell (see Non-Patent Documents 20, 21 and 22, and Patent Document 1), but the production amount is only around 1 to 15 μg/ml. In these cases, any of a metallothionein promoter a Rous sarcoma virus LTR promoter, and an adenovirus 2 major late promoter is used and, as a selectable marker, any of an aminoglycoside 3′ phosphotransferase (neo) gene, a dihydrofolate reductase (dhfr) gene and a histidinol resistant gene, or a combination thereof is used. In any case, a method for independently constructing each of expression vectors of genes each encoding an α chain, a β chain or a γ chain, transfecting a cell with three of them simultaneously, or transforming a cell with each two expression vectors having an α chain gene, a γ chain gene or a β chain gene and a γ chain gene in advance and, thereafter, introducing an expression vector having a β chain gene and an α chain gene, or a method for mixing an equal amount of a plasmid having an α chain gene and a γ chain gene and a plasmid having a β chain gene, and introducing the mixture into a cell is adopted. In any case, there is no particular description regarding a constitutional ratio of the respective genes upon introduction, and it is presumed that an equal amount of the respective genes are introduced as in the conventional procedure. In a medicament using blood-derived fibrinogen which is currently used, for example, in the case of a fibrin paste preparation, about 80 mg/dose of fibrinogen is used, and a manufacturing facility must be unavoidably scaled up at an expression amount of around ten and a few μg/ml as described above, and this inevitably leads to the high cost. For preparing fibrinogen at a practical level by the gene recombination technique, a highly producing cell (e.g., an expression amount of fibrinogen is 100 μg/ml or more) is necessary, but currently, there is no report of an expression system using a recombinant animal cell satisfying this.    Patent Document 1: U.S. Pat. No. 6,037,457    Non-Patent Document 1: “Apoptosis and Disease, Chapter; Central Nervous System Disease” edited by Yoshikuni Mizuno, Medicine and Drug Journal (2000)    Non-Patent Document 2: Tey B T et al., Biotechnol. Bioeng., 68, 31 (2000)    Non-Patent Document 3: Simpson N H et al., Biotechnol. Bioeng., 64, 174 (1999)    Non-Patent Document 4: Kim N S and Lee G M, Biotechnol. Bioeng., 82, 872 (2003)    Non-Patent Document 5: Kim N S and Lee G M, Biotechnol. Bioeng., 71, 184 (2000/2001)    Non-Patent Document 6: Kim N S and Lee G M, J. Biotechnol., 95, 237 (2002)    Non-Patent Document 7: O'Reilly L A et al., EMBO J., 15, 6979 (1996)    Non-Patent Document 8: Yang T et al., J Cell Physiol., 166, 523 (1996)    Non-Patent Document 9: Zhou Y et al., Am. J. Physiol. Endocrinol. Metab., 278, E340 (2000)    Non-Patent Document 10: Lakics V et al., J. Immuno., 165, 2729 (2000)    Non-Patent Document 11: Mercille S et al., Biotechnol. Bioeng., 63, 516 (1999)    Non-Patent Document 12: “Hemostasis, Thrombus, Fibrinogenolysis” edited by Matsuda and Suzuki, Chugai Igakusha (1994)    Non-Patent Document 13: Chung D E and Davie E W, Biochemistry, 23, 4232 (1984)    Non-Patent Document 14: Lawrence Y F et al., Biochemistry, 31, 11968 (1992)    Non-Patent Document 15: “Special Edition; Bioadhesive” Biomedical Perspectives, 6, 9-72 (1997)    Non-Patent Document 16: Redman C M and Kudryk B, J. Biol. Chem., 274, 554 (1999)    Non-Patent Document 17: T. Morita et al.: J. Biochemistry, 83, 559-570, (1978)    Non-Patent Document 18: Farrell D H et al., Biochemistry, 30, 9414 (1991)    Non-Patent Document 19: Roy S N et al., J. Biol. Chem., 266, 4758 (1991)    Non-Patent Document 20: Lord S T et al., Blood Coagul Fibrinolysis, 4, 55 (1993)    Non-Patent Document 21: Binnie C G et al., Biochemistry, 32, 107 (1993)    Non-Patent Document 22: Lord S T et al., Biochemistry. 35, 2342 (1996)